Browsing by Author "Holt, SA"
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- Item2nd Asia-Oceania Instrument Scientist Workshops, Manly, July 19, 2015(Taylor & Francis Online, 2015-11-17) Hester, JR; Holt, SA; Imperia, P; Piltz, RO; Rehm, C; Rule, KC; Mole, RA; McIntyre, GJNo abstract available
- ItemAdvancing the reflectometry cause at ANSTO - updates and upgrades to the time-of-flight Platypus Neutron Reflectometer.(International Conference on Neutron Scattering, 2017-07-12) Nelson, A; Holt, SA; Darmann, F; Klose, FSince the first suite of neutron scattering instruments was commissioned in 2008 the Australian Nuclear Science and Technology Organisation (ANSTO) has invested in instrumentation for the analysis of thin interfacial films. The horizontal time-of-flight reflectometer, Platypus , has now been joined by an X-ray reflectometer and a variable angle spectroscopic imaging ellipsometer. The high quality science possible on these complementary instruments has lead to a large oversubscription rate on Platypus. Here, we outline the key developments and upgrades we have made to Platypus that have led to this success. These include the development of event mode acquisition for studying kinetic processes, new sample environments (confinement cell, vapour delivery systems), as well as projects to upgrade its performance by installing new collimation systems and detectors.
- ItemAsymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic(The Royal Society, 2013-10-16) Clifton, LA; Skoda, MWA; Daulton, E; Hughes, AV; Le Brun, AP; Lakey, JH; Holt, SAThe Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo-like conditions. © 2013, The Royal Society.
- ItemBilayer-mediated clustering and functional interaction of MscL channels(Cell Press, 2011-03-02) Grage, SL; Keleshian, AM; Turdzeladze, T; Battle, AR; Tay, WC; May, RP; Holt, SA; Contera, SA; Haertlein, M; Moulin, M; Pal, P; Rohde, PR; Forsyth, VT; Watts, A; Huang, KC; Ulrich, AS; Martinac, BMechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.© 2011, Cell Press
- ItemCanted magnetism in modulated thin-film superlattices Oliver(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Paull, O; Wong., J; Sando, D; Lee, WT; Holt, SA; Valanoor, NInteractions at interfaces of magnetic materials such as giant magnetoresistance (GMR) form the physical foundation of many technological devices in today’s market. Although GMR technologies are now somewhat dated, interactions at the interface between layers of oxide materials continue to demonstrate attractive mechanisms for technology applications due to the wide variety of available oxide materials as well as their ability to be well lattice-matched to form heterostructures. Magnetic thin-film superlattices made of 15 repetitions of La2/3Sr1/3MnO3 (LSMO) and LaNiO3 (LNO) have been shown to exhibit a magnetic exchange interaction in LNO that is dependent upon its thickness n (where n is the number of unit cells of LNO) between LSMO layers . At n = 1, the LSMO layers are ferromagnetically aligned with eachother. At n = 3 however, the LSMO layers are canted by 110◦ with respect to one another. This canting is reportedly driven by an emergent c-axis spin-helix in LNO that arises due to charge transfer at the interfaces between LNO and LSMO . We have fabricated superlattices of similar quality to Ref.  using Reflected High Energy Electron Diffraction (RHEED) assisted Pulsed Laser Deposition (PLD), where RHEED is used to ensure unit-cell precision in layer thicknesses. These superlattices have been designed so they exhibit n = 3 and n = 1 interactions within the same superlattice in a modulated pattern. This modulation of the effective LNO exchange interaction in these samples creates a toy model of coupled magnetic layers to explore. The modulated design of these samples create “frustrated” environments where the effective exchange interaction is different on each side of an LSMO layer within the superlattice. We utilise polarised neutron reflectometry using PLAYPUS to reveal the complex magnetic profile of these modulated superlattices by measuring the non-spin-flip (R++, R−−), and spin-flip (R+−, R−+) cross sections. Presented are the current progress and prospects in the fitting process of these complex datasets. © The authors.
- ItemCharacterization of the seposition of n-Octanohydroxamate on copper surfaces(ECS, 2012-10) Parker, GK; Holt, SAThe interaction of n-octanohydroxamate with a copper electrode was investigated using cyclic voltammetry, contact angles, surface-enhanced Raman scattering, neutron reflectometry and X-ray reflectometry. The data demonstrated that hydroxamate specifically adsorbed at underpotentials in the keto tautomeric form, but the layer was hydrophilic and contained >50% water. In the CuI stability region the hydroxamate moiety took on the enol tautomeric form but the interface remained hydrated. Co-adsorption of electrolyte species was observed at underpotentials. The adsorbed hydroxamate at the surface did not prevent the formation of oxide in the CuI stability region. A hydrophobic non-passivating multilayer of copper n-octanohydroximate formed in the CuII stability region. © 2014, The Electrochemical Society.
- ItemDevelopments on the platypus neutron reflectometer(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Nelson, A; Le Brun, AP; Huang, TY; Paul, O; Holt, SAPLATYPUS is the initial neutron reflectometer at the Australian Centre for Neutron Scattering with the capability to study surface and interface systems ranging from biomolecules, soft matter through to magnetic thin films [1-3]. There have been a number of significant improvements to both the instrument and data reduction and treatment software  over the last two years. On the hardware front the original detector has been replaced yielding higher count-rate capabilities, greater detection efficiency at shorter wavelengths and significantly lower background. The slits which define the neutron beam have been replaced with upgraded positioning mechanisms enabling greater flexibility in experimental setup. These changes have significantly enhanced the instrument performance with improved reproducibility. This presentation will highlight the enhancements and recent publications.
- ItemDye⋯TiO2 interfacial structure of dye-sensitised solar cell working electrodes buried under a solution of I−/I3− redox electrolyte(Royal Society of Chemistry, 2017-07-27) McCree-Grey, J; Cole, JM; Holt, SA; Evans, PJ; Gong, YDye-sensitised solar cells (DSCs) have niche prospects for electricity-generating windows that could equip buildings for energy-sustainable future cities. However, this ‘smart window’ technology is being held back by a lack of understanding in how the dye interacts with its device environment at the molecular level. A better appreciation of the dye⋯TiO2 interfacial structure of the DSC working electrodes would be particularly valuable since associated structure–function relationships could be established; these rules would provide a ‘toolkit’ for the molecular engineering of more suitable DSC dyes via rational design. Previous materials characterisation efforts have been limited to determining this interfacial structure within an environment exposed to air or situated in a solvent medium. This study is the first to reveal the structure of this buried interface within the functional device environment, and represents the first application of in situ neutron reflectometry to DSC research. By incorporating the electrolyte into the structural model of this buried interface, we reveal how lithium cations from the electrolyte constituents influence the dye⋯TiO2 binding configuration of an organic sensitiser, MK-44, via Li+ complexation to the cyanoacrylate group. This dye is the molecular congener of the high-performance MK-2 DSC dye, whose hexa-alkyl chains appear to stabilise it from Li+ complexation. Our in situ neutron reflectometry findings are built up from auxiliary structural models derived from ex situ X-ray reflectometry and corroborated via density functional theory and UV/vis absorption spectroscopy. Significant differences between the in situ and ex situ dye⋯TiO2 interfacial structures are found, highlighting the need to characterise the molecular structure of DSC working electrodes while in a fully assembled device. © Royal Society of Chemistry 2020
- ItemExamining the creation and destruction of model bacterial surfaces(International Conference on Neutron Scattering, 2017-07-12) Clifton, LA; Skoda, MWA; Hughes, A; Holt, SA; Lakey, JBacteria are differentiated into two main groups, Gram-positive or Gram-negative, based on the Gram stain which detects the thick peptidoglycan cell wall of gram positive bacteria.Gram-negative bacteria are of particular biomedical and technological interest due to their role in disease, the increasing antibiotic resistance if some species and their utility in many biotechnological processes. The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS is a polyanionic molecule, with numerous phosphate groups present in the Lipid A and core oligosaccharide regions. We have attempted to create GNB-OM assays which are amenable to molecular level characterisation. These systems are asymmetric phospholipid : lipopolysaccharide membranes deposited at the solid/liquid interface and consist of either solid supported bilayers at the silicon/water interface or floating supported bilayers at the gold/water interface. The analysis of these membrane models by neutron reflectometry has provided new insights into the OM and interactions with it. Examples of this include providing conformation of the activity of anti-bacterial proteins, the role of the lipopolysaccharide polysaccharide chains in protecting the bacterium and the importance of divalent cations in stabilising the OM structure.
- ItemHigh coverage fluid-phase floating lipid bilayers supported by ω-thiolipid self-assembled monolayers(The Royal Society Publishing, 2014-09-06) Hughes, AV; Holt, SA; Daulton, E; Soliakov, A; Charlton, TR; Roser, SJ; Lakey, JHLarge area lipid bilayers, on solid surfaces, are useful in physical studies of biological membranes. It is advantageous to minimize the interactions of these bilayers with the substrate and this can be achieved via the formation of a floating supported bilayer (FSB) upon either a surface bound phospholipid bilayer or monolayer. The FSB's independence is enabled by the continuous water layer (greater than 15 Å) that remains between the two. However, previous FSBs have had limited stability and low density. Here, we demonstrate by surface plasmon resonance and neutron reflectivity, the formation of a complete self-assembled monolayer (SAM) on gold surfaces by a synthetic phosphatidylcholine bearing a thiol group at the end of one fatty acyl chain. Furthermore, a very dense FSB (more than 96%) of saturated phosphatidylcholine can be formed on this SAM by sequential Langmuir–Blodgett and Langmuir–Schaefer procedures. Neutron reflectivity used both isotopic and magnetic contrast to enhance the accuracy of the data fits. This system offers the means to study transmembrane proteins, membrane potential effects (using the gold as an electrode) and even model bacterial outer membranes. Using unsaturated phosphatidylcholines, which have previously failed to form stable FSBs, we achieved a coverage of 73%. © 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.
- ItemHot commissioning and first user experiments on the Spatz neutron reflectometer(Australian Nuclear Science and Technology Organisation, 2021-11-26) Le Brun, AP; Huang, TY; Pullen, SA; Nelson, A; Holt, SAThe Spatz neutron beam instrument is the latest to be installed and commissioned in the Neutron Guide Hall at the 20 MW OPAL Research Reactor. Spatz is a time-of-flight neutron reflectometer used for studying nanoscale structures at surfaces and interfaces and utilises a vertical sample geometry / horizontal scattering geometry. The instrument is situated at the end position of the CG2B neutron guide and views the cold neutron source (CNS). The disc chopper cascade that pulses the neutron beam to produce the time-of-flight is very configurable to provide a wavelength resolution between 1 to 12 %. The detector is a helium-3 two dimensional detector that is capable of measuring both specular and off-specular reflectivity. The sample stage can support a range of different sample environments including multiple solid-liquid cells, an atmospheric chamber with temperature control, the ATR-FT-IR spectrometer for simultaneous infra-red spectroscopy and neutron reflectometry measurements, electrochemical cells, etc. The geometry of the instrument and the sample environment available means that Spatz is well suited to studying phenomena at the gas-solid interface and solid-liquid interface. The Spatz instrument has been fully commissioned with neutrons and the results of the commissioning are presented. This includes measurements using the ‘Bragg mirror’ consisting of 25 bilayers of nickel and titanium, different solid substrates of silicon, quartz and sapphire, spin-coated polymer samples, and films under liquid. Reflectivity down to 10-7 can be achieved within 1 hour measuring time with good counting statistics in most cases. Early user experiments cover a range of science including investigating the thermal stability of organic solar cell materials and proteins interacting with biomimetic phospholipid cell membranes. © 2021 The Authors
- ItemThe impact of pH on packing in tethered lipid bilayers(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Cranfield, CG; Berry, T; Holt, SA; Le Brun, AP; Valenzuela, SM; Coster, HGL; Cornell, BAWe report that increasing the H3O+ concentration when lowering the pH reduces the intrinsic ionic conduction through phospholipid bilayers (Fig 1A), which is counter to what might be expected from increasing the H3O+ concentration. We attribute the conduction decrease to a reduction of the molecular area per lipid (ao). These effects are seen at H3O+ concentrations in the range nM to µM despite these being very low concentrations compared to that of a typical bathing electrolyte solution of 135mM ionic concentration. We present a model, in which the pH dependent reduction in ao favours an increase in lipid packing. To support this model, we provide evidence of the effects of the hydronium ion on lipid geometry using neutron reflectometry (Fig 1C). Further examples will be given of the impact of the H3O ion concentration on the hydrogen bonding within the polar groups of lipid.
- ItemIn situ study of the impact of acidic and neutral deposition pH on alkane phosphate film formation and stability on TiO2(Royal Society Chemistry, 2013-02-28) Holt, SA; Le Brun, AP; Nelson, A; Lakey, JHTiO2 has been used as a model system for the surface of medical implant devices based upon titanium alloys. Self Assembled Monolayers (SAM) can be applied via a specific phosphate interaction to modify the surface properties of the TiO2 substrate. Here it is demonstrated via in situ Quartz Crystal Microbalance and Neutron Reflectometry experiments that the deposition of dodecyl phosphate onto TiO2 proceeds more rapidly at pH 4.5 than at pH 7.0. Conversely, the film stability was enhanced for films deposited at pH 7.0, demonstrating that while the initial association with the surface is driven by electrostatics it does not determine the SAM density. While the adsorbed amount appeared to be relatively constant after a few minutes incubation time it was found that washing with buffer removed about 50% of the adsorbed material after these short incubation times. With incubation time of the order of hours the proportion of the film washed off the surface decreased demonstrating that the specific phosphate-TiO2 interaction was a slow process. The slower initial surface interaction at pH 7.0 therefore allowed greater re-arrangement of the dodecyl phosphate resulting in more complete and robust monolayers than at pH 4.5. This was demonstrated by washing the film with buffer of increasing pH of up to 9.5. For SAM longevity on titanium alloys it is clear that slow deposition at pH 7 produces more robust films than rapid deposition at lower pH values.© 2013, Royal Society of Chemistry.
- ItemAn in vitro model to investigate the interactions between antimicrobial peptides and the outer membrane of gram-negative pathogens(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Han, ML; Shen, HH; Zhu, Y; Le Brun, AP; Holt, SA; Roberts, K; Song, JN; Cooper, MA; Moskowitz, SM; Velkov, T; Li, JIncreasing antibiotic resistance in Gram-negative bacteria led to polymyxins as the last therapy. Polymyxins present their antimicrobial activity through an initial electronical interaction with lipid A in the outer membrane (OM) of GNB, and the most common mechanism of polymyxin resistance is through modifications of lipid A with positively charged groups, such as 4-amino-L-arabinose (L-Aar4N) or phosphoethanolamine (pEtN). However, it is notable that Gram-negative bacteria employ a combination of charge-charge repulsion mechanism and the modification to fatty acyl chains of lipid A to obtain high-level polymyxin resistance. Hence, we designed hydrophobic polymyxin-related lipopeptides in order to overcome modified lipid A to insert into the outer membrane of Gram-negative bacteria. In this study, we employed neutron reflectometry (NR) study to investigate the interactions between lipid A and polymyxins. Lipid A was extracted from polymyxin-susceptible and -resistant pseudomonas aeruginosa strains, and analysed using ESI-MS in the negative ion mode. The asymmetric lipid A: deuterated DPPC bilayers were deposited on SiO2 surfaces by combined Langmuir-Blodgett and Langmuir-Schaefer disposition methods, and characterised by neutron reflectometer. Our results showed L-Ara4N modified lipid A was observed in polymyxin-resistant PAKpmrB6 strain, but not in the wild-type PAK strain. The NR data obtained from unmodified lipid A: DPPC bilayer was fitted into a five-layer model. Whereas, a six-layer model containing an extra outer headgroup was established for L-Ara4N modified lipid A: d-DPPC bilayer. Our results showed a dense of PMB (volume fraction of >20%) bound to the surface of both unmodified and modified lipid A: DPPC bilayers. While it is notable that the significant changes in NR profiles obtained from H2O contrast indicated about 15.8% and 6.1% of PMB penetrated into the wild-type lipid A headgroup and fatty acyl chains, respectively, but without penetration into L-Ara4N-lipid A: d-DPPC bilayer. However, the employment of octpeptin A3 induced higher hydrophobic interactions with L-Ara4N-lipid A: d-DPPC bilayer. Our study provides an in vitro model to investigate the interactions of polymyxins with OM bilayers in GNB, and confirmed that lipid A modification with L-Ara4N was certainly to reduce the penetration of PMB into bacterial membranes. Remarkably, the higher binding affinity between octapeptin A3 and L-Ara4N modified lipid A indicated its potential to be the new generation antibiotics for the therapy of infections caused by multi-drug resistant Gram negative bacteria.
- ItemIon irradiation as a tool for modifying the surface and optical properties of plasma polymerised thin films(Elsevier B.V., 2015-10-01) Grant, DS; Bazaka, K; Siegele, R; Holt, SA; Jacob, MVRadio frequency (R.F.) glow discharge polyterpenol thin films were prepared on silicon wafers and irradiated with I10+ ions to fluences of 1 × 1010 and 1 × 1012 ions/cm2. Post-irradiation characterisation of these films indicated the development of well-defined nano-scale ion entry tracks, highlighting prospective applications for ion irradiated polyterpenol thin films in a variety of membrane and nanotube-fabrication functions. Optical characterisation showed the films to be optically transparent within the visible spectrum and revealed an ability to selectively control the thin film refractive index as a function of fluence. This indicates that ion irradiation processing may be employed to produce plasma-polymer waveguides to accommodate a variety of wavelengths. XRR probing of the substrate-thin film interface revealed interfacial roughness values comparable to those obtained for the uncoated substrate’s surface (i.e., both on the order of 5 Å), indicating minimal substrate etching during the plasma deposition process. © 2015, Elsevier B.V.
- ItemThe multipurpose time-of-flight neutron reflectometer "Platypus" at Australia's OPAL reactor(Elsevier Science BV, 2011-03-11) James, M; Nelson, A; Holt, SA; Saerbeck, T; Hamilton, WA; Klose, FIn this manuscript we describe the major components of the Platypus time-of-flight neutron reflectometer at the 20 MW OPAL reactor in Sydney, Australia. Platypus is a multipurpose spectrometer for the characterisation of solid thin films, materials adsorbed at the solid-liquid interface and free-liquid surfaces. It also has the capacity to study magnetic thin films using spin-polarised neutrons. Platypus utilises a white neutron beam (lambda=2-20 angstrom) that is pulsed using boron-coated disc chopper pairs; thus providing the capacity to tailor the wavelength resolution of the pulses to suit the system under investigation. Supermirror optical components are used to focus, deflect or spin-polarise the broad bandwidth neutron beams, and typical incident spectra are presented for each configuration. A series of neutron reflectivity datasets are presented, indicating the quality and flexibility of this spectrometer. Minimum reflectivity values of <10(-7) are observed: while maximum thickness values of 325 nm have been measured for single-component films and 483 nm for a multilayer system. Off-specular measurements have also been made to investigate in-plane features as opposed to those normal to the sample surface. Finally, the first published studies conducted using the Platypus time-of-flight neutron reflectometer are presented. (C) 2011 Elsevier B.V. All rights reserved.
- ItemPolarization "down under": the polarized time-of-flight neutron reflectometer PLATYPUS(American Institute of Physics, 2012-08-01) Saerbeck, T; Klose, F; Le Brun, AP; Füzi, J; Brûlé, A; Nelson, A; Holt, SA; James, MThis review presents the implementation and full characterization of the polarization equipment of the time-of-flightneutron reflectometer PLATYPUS at the Australian Nuclear Science and Technology Organisation (ANSTO). The functionality and efficiency of individual components are evaluated and found to maintain a high neutron beam polarization with a maximum of 99.3% through polarizing Fe/Si supermirrors. Neutron spin-flippers with efficiencies of 99.7% give full control over the incident and scattered neutron spin direction over the whole wavelength spectrum available in the instrument. The first scientific experiments illustrate data correction mechanisms for finite polarizations and reveal an extraordinarily high reproducibility for measuring magnetic thin film samples. The setup is now fully commissioned and available for users through the neutron beam proposal system of the Bragg Institute at ANSTO. © 2012, American Institute of Physics.
- ItemProgress with hot commissioning and first user experiments on the SPATZ neutron reflectometer(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Le Brun, AP; Huang, TY; Nelson, A; Holt, SAThe SPATZ neutron reflectometer was originally the V18 BioRef reflectometer at the BER-II Research Reactor (Berlin, Germany) [1, 2], and was transferred from HZB to ANSTO ownership during October 2016 to February 2017. The reflectometer was re-installed at ANSTO’s 20 MW OPAL Research Reactor in the Neutron Guide Hall on the end position of the newly installed CG2B cold-neutron guide. The hot commissioning licence was issued in October 2018 with first neutrons in November 2018. Radiation surveys and shielding improvements were completed in May 2019. Since this time, hot commissioning has continued and has involved evaluating the performance of the instrument. SPATZ is a time-of-flight instrument, and the ability to translate chopper 2 makes the instrument highly configurable with the ability to select a time resolution from 1 to 12 %. A number of different sample environments have been tested including: solid-liquid cells with a HPLC pump for solvent contrast exchange, circulating water baths for temperature control, and the infrared spectrometer for simultaneous neutron reflectometry and ATR-FT-IR spectroscopy. The operating license was issued in December 2019, and first experiments are scheduled for the last quarter of 2020. SPATZ is now available for user proposals in the ACNS portal . This presentation will highlight the instrument performance and achievements during hot commissioning and give an overview of the user program to date and first data collected.
- ItemReconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes(Macmillan Publishers Limited., 2014-10-24) Shen, HH; Leyton, DL; Shiota, T; Belousoff, MJ; Noinaj, N; Lu, J; Holt, SA; Tan, K; Selkrig, J; Webb, CT; Buchanan, SK; Martin, LL; Lithgow, TIn biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines. © Macmillan Publishers Limited
- ItemRegulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol(Public Library of Science, 2013-02-14) Valenzuela, SM; Alkhamici, H; Brown, LJ; Almond, OC; Goodchild, SC; Carne, S; Curmi, PMG; Holt, SA; Cornell, BAThe Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer. © 2013, Valenzuela et al.